Methods for diagnosing and treating diseases and conditions associated with protein kinase Cλ

ABSTRACT

The invention provides methods of diagnosing diseases and conditions associated with PKCλ, methods for identifying compounds that can be used to treat or to prevent such diseases and conditions, and methods of using these compounds to treat or to prevent such diseases and conditions. Also provided in the invention are animal model systems that can be used in screening methods.

This application is a U.S. National Stage application of, and claimspriority under 35 U.S.C. §371 from, International Application No.PCT/US02/28410, filed on Sep. 6, 2002, and claims priority from U.S.Ser. No. 60/317,653, filed on Sep. 6, 2001.

FIELD OF THE INVENTION

This invention relates to methods for diagnosing and treating diseasesand conditions associated with Protein Kinase C λ.

BACKGROUND OF THE INVENTION

The processes by which organs acquire global structures and patternsduring development are highly complex, and likely involve multiple,overlapping biochemical pathways. In the vertebrate heart, for example,the first key visible step in this process is chamber morphogenesis,involving the fashioning of the atrium and the ventricle. Properorientation of these two functionally distinct contractile units isrequired for unidirectional blood flow, which begins with the firstheartbeat of an organism. Properly formed chambers thereafter are thesubstrates upon which further heart development is superimposed.

Over recent years, much has been learned about the molecular mechanismsthat are responsible for the acquisition of characteristic atrial andventricular cell fates (Fishman et al., Development 124:2099-2117, 1997;Srivastava et al., Nature 407:221-226, 2000). However, bothembryological and molecular steps that fashion the higher orderstructures of these chambers have proven to be more elusive because, inpart, unlike cell fate decisions, these steps can be studiedmeaningfully only in living organisms. The zebrafish, Danio rerio, is aconvenient organism to use in genetic and biochemical analyses ofdevelopment. It has an accessible and transparent embryo, allowingdirect observation of organ function from the earliest stages ofdevelopment, has a short generation time, and is fecund.

SUMMARY OF THE INVENTION

The invention provides diagnostic, drug screening, and therapeuticmethods that are based on the observation that a mutation, designatedthe “heart and soul (has)” mutation, in the zebrafish Protein Kinase C λ(PKCλ) gene, as well as a small molecule identified in a chemical screenin zebrafish, concentramide, cause abnormal heart growth anddevelopment.

In a first aspect, the invention provides a method of determiningwhether a test subject (e.g., a mammal, such as a human) has or is atrisk of developing a disease or condition related to PKCλ (e.g., adisease or condition of the heart; also see below). This method involvesanalyzing a nucleic acid molecule of a sample from the test subject todetermine whether the test subject has a mutation (e.g., the hasmutation; see below) in a gene encoding PKCλ. The presence of such amutation indicates that the test subject has or is at risk of developinga disease related to PKCλ. This method can also involve the step ofusing nucleic acid molecule primers specific for a gene encoding PKCλfor nucleic acid molecule amplification of the gene by the polymerasechain reaction. It can further involve sequencing a nucleic acidmolecule encoding PKCλ from a test subject.

In a second aspect, the invention provides a method for identifyingcompounds that can be used to treat or prevent a disease or conditionassociated with PKCλ, or in the preparation of a medicament for use insuch methods. This method involves contacting an organism (e.g., azebrafish) having a mutation in a PKCλ gene (e.g., the heart and soulmutation), and having a phenotype characteristic of such a disease orcondition, with the compound, and determining the effect of the compoundon the phenotype. Detection of an improvement in the phenotype indicatesthe identification of a compound that can be used to treat or preventthe disease or condition. In a variation of this method, the organism,with or without a mutation in the PKCλ gene (e.g., the has mutation), iscontacted with a candidate compound in the presence of concentramide.

In a third aspect, the invention provides a method of treating orpreventing a disease or condition related to PKCλ in a patient (e.g., apatient having a mutation (e.g., the heart and soul mutation) in a PKCλgene), involving administering to the patient a compound identifiedusing the method described above. Also included in the invention is theuse of such compounds in the treatment or prevention of such diseases orconditions, as well as the use of these compounds in the preparation ofmedicaments for such treatment or prevention.

In a fourth aspect, the invention provides an additional method oftreating or preventing a disease or condition related to PKCλ in apatient. This method involves administering to the patient a functionalPKCλ protein or a nucleic acid molecule (in, e.g., an expression vector)encoding the protein. Also included in the invention is the use of suchproteins or nucleic acid molecules in the treatment or prevention ofsuch diseases or conditions, as well as the use of these proteins ornucleic acid molecules in the preparation of medicaments for suchtreatment or prevention.

In a fifth aspect, the invention includes a substantially pure zebrafishPKCλ polypeptide. This polypeptide can include or consist essentiallyof, for example, an amino acid sequence that is substantially identicalto the amino acid sequence of SEQ ID NO:2. The invention also includesvariants of these polypeptides that include sequences that are at least75%, 85%, or 95% identical to the sequences of these polypeptides, andwhich have PKCλ activity or otherwise are characteristic of the diseasesand conditions mentioned elsewhere herein. Fragments of thesepolypeptides are also included in the invention. For example, fragmentsthat include any of the different domains of PKCλ, in varyingcombinations, are included.

In a sixth aspect, the invention provides an isolated nucleic acidmolecule (e.g., a DNA molecule) including a sequence encoding azebrafish PKCλ polypeptide. This nucleic acid molecule can encode apolypeptide including or consisting essentially of an amino sequencethat is substantially identical to the amino acid sequence of SEQ IDNO:2. The invention also includes nucleic acid molecules that hybridizeto the complement of SEQ ID NO:1 under highly stringent conditions andencode polypeptides that have PKCλ activity or otherwise arecharacteristic of the diseases and conditions mentioned elsewhereherein.

In a seventh aspect, the invention provides a vector including thenucleic acid molecule described above.

In an eighth aspect, the invention includes a cell including the vectordescribed above.

In a ninth aspect, the invention provides a non-human transgenic animal(e.g., a zebrafish or a mouse) including the nucleic acid moleculedescribed above.

In a tenth aspect, the invention provides a non-human animal having aknockout mutation in one or both alleles encoding a PKCλ polypeptide.

In an eleventh aspect the invention includes a cell from the non-humanknockout animal described above.

In a twelfth aspect, the invention includes a non-human transgenicanimal (e.g., a zebrafish) including a nucleic acid molecule encoding amutant PKCλ polypeptide, e.g., a polypeptide having the heart and soulmutation.

In a thirteenth aspect, the invention provides an antibody thatspecifically binds to a PKCλ polypeptide.

By “polypeptide” or “polypeptide fragment” is meant a chain of two ormore (e.g., 10, 15, 20, 30, 50, 100, or 200, or more) amino acids,regardless of any post-translational modification (e.g., glycosylationor phosphorylation), constituting all or part of a naturally ornon-naturally occurring polypeptide. By “post-translationalmodification” is meant any change to a polypeptide or polypeptidefragment during or after synthesis. Post-translational modifications canbe produced naturally (such as during synthesis within a cell) orgenerated artificially (such as by recombinant or chemical means). A“protein” can be made up of one or more polypeptides.

By “Protein Kinase C λ protein,” “Protein Kinase C λ polypeptide,” “PKCλprotein,” or “PKCλ polypeptide” is meant a polypeptide that has at least45%, preferably at least 60%, more preferably at least 75%, and mostpreferably at least 90% amino acid sequence identity to the sequence ofa human (SEQ ID NO:5) or a zebrafish (SEQ ID NO:2) PKCλ polypeptide.Polypeptide products from splice variants of PKCλ gene sequences andPKCλ genes containing mutations are also included in this definition. APKCλ polypeptide as defined herein plays a role in heart development,modeling, and function. It can be used as a marker of diseases andconditions associated with PKCλ, such as heart disease (also see below).

By a “Protein Kinase C λ nucleic acid molecule” or “PKCλ nucleic acidmolecule” is meant a nucleic acid molecule, such as a genomic DNA, cDNA,or RNA (e.g., mRNA) molecule, that encodes a PKCλ protein (e.g., a human(encoded by SEQ ID NO:4) or a zebrafish (encoded by SEQ ID NOs:1 or 3)PKCλ protein), a PKCλ polypeptide, or a portion thereof, as definedabove. A mutation in a PKCλ nucleic acid molecule can be characterized,for example, by the insertion of a premature stop codon anywhere in thePKCλ gene. For example, codon R515 can be changed to a stop codon (CGAto TGA), or codon W519 can be changed to a stop codon (TGG to TAG). Inaddition to this zebrafish Protein Kinase C λ mutation (hereinafterreferred to as “the heart and soul mutation”), the invention includesany mutation that results in aberrant PKCλ protein production orfunction, including, only as examples, null mutations and additionalmutations causing truncations. The truncations can be carboxyl terminaltruncations in which the carboxyl terminal half of the protein (or aportion thereof) is not produced. For example, at least 10, 25, 50, 70,75, 100, 150, 200, or 250 amino acids of the carboxyl terminal half ofthe protein can be absent.

The term “identity” is used herein to describe the relationship of thesequence of a particular nucleic acid molecule or polypeptide to thesequence of a reference molecule of the same type. For example, if apolypeptide or a nucleic acid molecule has the same amino acid ornucleotide residue at a given position, compared to a reference moleculeto which it is aligned, there is said to be “identity” at that position.The level of sequence identity of a nucleic acid molecule or apolypeptide to a reference molecule is typically measured using sequenceanalysis software with the default parameters specified therein, such asthe introduction of gaps to achieve an optimal alignment (e.g., SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705, BLAST, or PILEUP/PRETTYBOX programs). These software programsmatch identical or similar sequences by assigning degrees of identity tovarious substitutions, deletions, or other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine, valine, isoleucine, and leucine; asparticacid, glutamic acid, asparagine, and glutamine; serine and threonine;lysine and arginine; and phenylalanine and tyrosine.

A nucleic acid molecule or polypeptide is said to be “substantiallyidentical” to a reference molecule if it exhibits, over its entirelength, at least 51%, preferably at least 55%, 60%, or 65%, and mostpreferably 75%, 85%, 90%, or 95% identity to the sequence of thereference molecule. For polypeptides, the length of comparison sequencesis at least 16 amino acids, preferably at least 20 amino acids, morepreferably at least 25 amino acids, and most preferably at least 35amino acids. For nucleic acid molecules, the length of comparisonsequences is at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably at least 110 nucleotides. Of course, the length of comparisoncan be any length up to and including full length.

A PKCλ nucleic acid molecule or a PKCλ polypeptide is “analyzed” orsubject to “analysis” if a test procedure is carried out on it thatallows the determination of its biological activity or whether it iswild type or mutated. For example, one can analyze the PKCλ genes of ananimal (e.g., a human or a zebrafish) by amplifying genomic DNA of theanimal using the polymerase chain reaction, and then determining whetherthe amplified DNA contains a mutation, for example, the heart and soulmutation, by, e.g., nucleotide sequence or restriction fragmentanalysis.

By “probe” or “primer” is meant a single-stranded DNA or RNA molecule ofdefined sequence that can base pair to a second DNA or RNA molecule thatcontains a complementary sequence (a “target”). The stability of theresulting hybrid depends upon the extent of the base pairing thatoccurs. This stability is affected by parameters such as the degree ofcomplementarity between the probe and target molecule, and the degree ofstringency of the hybridization conditions. The degree of hybridizationstringency is affected by parameters such as the temperature, saltconcentration, and concentration of organic molecules, such asformamide, and is determined by methods that are well known to thoseskilled in the art. Probes or primers specific for PKCλ nucleic acidmolecules, preferably, have greater than 45% sequence identity, morepreferably at least 55-75% sequence identity, still more preferably atleast 75-85% sequence identity, yet more preferably at least 85-99%sequence identity, and most preferably 100% sequence identity to thesequences of human (SEQ ID NO:4) or zebrafish (SEQ ID NOs:1 and 3) PKCλgenes.

Probes can be detectably labeled, either radioactively ornon-radioactively, by methods that are well known to those skilled inthe art. Probes can be used for methods involving nucleic acidhybridization, such as nucleic acid sequencing, nucleic acidamplification by the polymerase chain reaction, single strandedconformational polymorphism (SSCP) analysis, restriction fragmentpolymorphism (RFLP) analysis, Southern hybridization, northernhybridization, in situ hybridization, electrophoretic mobility shiftassay (EMSA), and other methods that are well known to those skilled inthe art.

A molecule, e.g., an oligonucleotide probe or primer, a gene or fragmentthereof, a cDNA molecule, a polypeptide, or an antibody, can be said tobe “detectably-labeled” if it is marked in such a way that its presencecan be directly identified in a sample. Methods for detectably labelingmolecules are well known in the art and include, without limitation,radioactive labeling (e.g., with an isotope, such as ³²P or ³⁵S) andnonradioactive labeling (e.g., with a fluorescent label, such asfluorescein).

By a “substantially pure polypeptide” is meant a polypeptide (or afragment thereof) that has been separated from proteins and organicmolecules that naturally accompany it. Typically, a polypeptide issubstantially pure when it is at least 60%, by weight, free from theproteins and naturally occurring organic molecules with which it isnaturally associated. Preferably, the polypeptide is a PKCλ polypeptidethat is at least 75%, more preferably at least 90%, and most preferablyat least 99%, by weight, pure. A substantially pure PKCλ polypeptide canbe obtained, for example, by extraction from a natural source, byexpression of a recombinant nucleic acid molecule encoding a PKCλpolypeptide, or by chemical synthesis. Purity can be measured by anyappropriate method, e.g., by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

A polypeptide is substantially free of naturally associated componentswhen it is separated from those proteins and organic molecules thataccompany it in its natural state. Thus, a protein that is chemicallysynthesized or produced in a cellular system that is different from thecell in which it is naturally produced is substantially free from itsnaturally associated components. Accordingly, substantially purepolypeptides not only include those that are derived from eukaryoticorganisms, but also those synthesized in E. coli, other prokaryotes, orin other such systems.

By “isolated nucleic acid molecule” is meant a nucleic acid moleculethat is removed from the environment in which it naturally occurs. Forexample, a naturally-occurring nucleic acid molecule present in thegenome of cell or as part of a gene bank is not isolated, but the samemolecule, separated from the remaining part of the genome, as a resultof, e.g., a cloning event (amplification), is “isolated.” Typically, anisolated nucleic acid molecule is free from nucleic acid regions (e.g.,coding regions) with which it is immediately contiguous, at the 5′ or 3′ends, in the naturally occurring genome. Such isolated nucleic acidmolecules can be part of a vector or a composition and still beisolated, as such a vector or composition is not part of its naturalenvironment.

An antibody is said to “specifically bind” to a polypeptide if itrecognizes and binds to the polypeptide (e.g., a PKCλ polypeptide), butdoes not substantially recognize and bind to other molecules (e.g.,non-PKCλ-related polypeptides) in a sample, e.g., a biological sample,which naturally includes the polypeptide.

By “high stringency conditions” is meant conditions that allowhybridization comparable with the hybridization that occurs using a DNAprobe of at least 100, e.g., 200, 350, or 500, nucleotides in length, ina buffer containing 0.5 M NaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA(fraction V), at a temperature of 65° C., or a buffer containing 48%formamide, 4.8×SSC, 0.2 M Tris-Cl, pH 7.6, 1× Denhardt's solution, 10%dextran sulfate, and 0.1% SDS, at a temperature of 42° C. (These aretypical conditions for high stringency northern or Southernhybridizations.) High stringency hybridization is also relied upon forthe success of numerous techniques routinely performed by molecularbiologists, such as high stringency PCR, DNA sequencing, single strandconformational polymorphism analysis, and in situ hybridization. Incontrast to northern and Southern hybridizations, these techniques areusually performed with relatively short probes (e.g., usually 16nucleotides or longer for PCR or sequencing, and 40 nucleotides orlonger for in situ hybridization). The high stringency conditions usedin these techniques are well known to those skilled in the art ofmolecular biology, and examples of them can be found, for example, inAusubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, N.Y., 1998, which is hereby incorporated by reference.

By “sample” is meant a tissue biopsy, amniotic fluid, cell, blood,serum, urine, stool, or other specimen obtained from a patient or a testsubject. The sample can be analyzed to detect a mutation in a PKCλ gene,or expression levels of a PKCλ gene, by methods that are known in theart. For example, methods such as sequencing, single-strandconformational polymorphism (SSCP) analysis, or restriction fragmentlength polymorphism (RFLP) analysis of PCR products derived from apatient sample can be used to detect a mutation in a PKCλ gene; ELISAand other immunoassays can be used to measure levels of a PKCλpolypeptide; and PCR can be used to measure the level of a PKCλ nucleicacid molecule.

By “Protein Kinase C λ-related disease,” “PKCλ-related disease,”“Protein Kinase C λ-related condition,” or “PKCλ-related condition” ismeant a disease or condition that results from inappropriately high orlow expression of a PKCλ gene, or a mutation in a PKCλ gene (includingcontrol sequences, such as promoters) that alters the biologicalactivity of a PKCλ nucleic acid molecule or polypeptide. PKCλ-relateddiseases and conditions can arise in any tissue in which PKCλ isexpressed during prenatal or post-natal life. PKCλ-related diseases andconditions can include diseases or conditions of the heart or cancer(also see below).

The invention provides several advantages. For example, using thediagnostic methods of the invention it is possible to detect anincreased likelihood of diseases or conditions associated with PKCλ,such as diseases of the heart or cancer, in a patient, so thatappropriate intervention can be instituted before any symptoms occur.This may be useful, for example, with patients in high-risk groups forsuch diseases or conditions. Also, the diagnostic methods of theinvention facilitate determination of the etiology of such an existingdisease or condition in a patient, so that an appropriate approach totreatment can be selected. In addition, the screening methods of theinvention can be used to identify compounds that can be used to treat orto prevent these diseases or conditions. The invention can also be usedto treat diseases or conditions (e.g., organ failure, such as heart orkidney failure) for which, prior to the invention, the only treatmentwas organ transplantation, which is limited by the availability of donororgans and the possibility of organ rejection.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the structure of a smallmolecule, concentramide, that alters heart patterning.

FIG. 1B is a lateral view of the mushroom-shaped heart of a live,concentramide-treated embryo 30 hpf. The atrium is indicated with A, andthe ventricle with V.

FIG. 1C is a schematic representation of a timecourse of concentramideeffectiveness. Black bars indicate the developmental time periods duringwhich groups of embryos were immersed in water containing concentramide.An “x” indicates that treatment during the indicated time period altersthe wild-type brain or heart phenotypes. An “o” indicates that thewild-type phenotype was observed. Blue and pink boxes mark the criticalperiods for development of the brain and heart phenotypes, respectively.

FIG. 2 shows that hearts from has mutant embryos phenocopy hearts fromconcentramide-treated embryos. In situ hybridization was performed withwild-type (FIGS. 2A-2C), concentramide-treated (FIGS. 2D-2F), and has(FIGS. 2G-2I) embryos. The expression pattern of cardiac myosin lightchain 2 (cmlc2) is shown for embryos 24 hpf (FIGS. 2A, 2D, and 2G) and30 hpf (FIGS. 2B, 2E, and 2H). The relative locations of atrium (A) andventricle (V) were confirmed by 7 μm sagital sections of embryos inwhich the ventricle was prestained blue by in situ hybridization toventricle-specific myosin heavy chain (vmhc), followed by staining ofthe atrium brown with the atrium-specific antibody S46 (FIGS. 2C, 2F,and 2I). The view is dorsal, anterior up in FIGS. 2A, 2D, and 2G. Theview is lateral, anterior to the left in all other frames.

FIG. 3A is a map of the has interval with genomic structure of thezebrafish PKCλ gene. YAC and BAC clones are indicated by addressesbeginning with “y” and “b.” The BAC clone 23c14 was sequenced todetermine the entire genomic structure of the has gene. From the partialsequence of the BACs listed, a preliminary transcript map of the regionwas determined (see Table 1). The zebrafish PKCλ gene comprises 18 exonsrepresented by vertical lines. The site of the mutations associated withthe m129 and m567 alleles is indicated with an asterisk.

FIG. 3B is an anti-PKCλ western blot of protein extracts from wild-typeembryos (WT), has mutant embryos (m567 −/−), and siblings of has mutantembryos (m567 +/+ and +/−).

FIGS. 3C-3E show that antisense disruption of PKCλ expressionphenocopies the has mutation. Wild-type embryos (3C), has embryos (3D),and wild-type embryos injected with a PKCλ antisense morpholino oligomer(3E) were photographed live 2 days postfertilization.

FIG. 4 shows that PKCλ is required for lamination, cell polarity, andepithelial cell-cell interaction in the retina. Transverse 5 μm sectionsof wild-type (FIGS. 4A-4B), concentramide-treated (FIGS. 4C-4D), and has(FIGS. 4E-4F) embryos were stained with hematoxylin-eosin 5 dayspostfertilization (FIGS. 4A, 4C, and 4E) or with dapi 30 hpf (FIGS. 4B,4D, and 4F). Arrowheads indicate mitotic nuclei. Zonula occludens-1localization in the retina is shown by 5 μm transverse sectionsfollowing staining of wild-type (FIG. 4G) or has (FIG. 4H) embryos withan anti-ZO-1 antibody.

FIG. 5 shows the effects of PKCλ inactivation and concentramidetreatment on polarity of the zebrafish kidney and the C. elegans embryo.An apical kidney marker (3G8) was used to stain kidneys of wild-type(FIG. 5A), concentramide-treated (FIG. 5B), and has (FIG. 5C) embryos.Transverse 2 μm sections of the pronephric duct are shown. FIGS. 5D and5E, C. elegans strain KK871, a stable expresser of a par2:GFP fusionprotein, was treated with 34 μM concentramide and allowed to develop atroom temperature. Nomarsid (FIG. 5D) and fluorescence (FIG. 5E)microscopy were used to visualize the asymmetry of division and par2:GFPlocalization after the first cell division. Posterior is to the left.

FIG. 6 shows alterations in anterior-posterior patterning aftertreatment with concentramide. FIGS. 6A-6C, In situ hybridization wasused to show Pax2.1 expression in untreated (FIG. 6A) andconcentramide-treated (FIG. 6B) 18-somite embryos. The expressionpatterns have been false-colored blue for untreated embryos and red forconcentramide-treated embryos. FIG. 6C shows an overlay of the imagesfrom FIGS. 6A and 6B. Arrowheads indicate areas of Pax2.1 expression atthe midbrain-hindbrain boundary and in the otic placodes. The view islateral, anterior to the left in FIGS. 6A-6C. FIG. 6D, The distancebetween the anterior edge of the heart field, as defined by cmlc2 insitu staining, and the rostral extreme of the zebrafish embryo wasmeasured in wild-type (WT), concentramide-treated (conc.), and hasembryos at the 18-somite stage. Error bars represent standard error.

FIG. 7 shows the order of anterior and posterior heart field fusion.Dorsal views of cmlc2 expression at the 16-somite (FIGS. 7A-7C) and18-somite (FIGS. 7D-7F) stages. Expression patterns for wild-type (FIG.7A and FIG. 7D), concentramide-treated (FIG. 7B and FIG. 7E), and has(FIGS. 7C and 7F) embryos are shown. Anterior is up.

FIG. 8 is a schematic representation of a model for chamber patterningin the zebrafish heart. Normally, the bilateral primordia of the heartfield converge and fuse first at the posterior end, followed by theanterior end to form a cone. The cone then rotates to orient atrialprecursors toward the anterior and ventricular precursors toward theposterior in an extended heart tube. In concentramide-treated and hasmutant embryos, the fusion order of the ends of the heart field isreversed, proceeding from the anterior to the posterior end. Rotation ofthe cone is blocked, preventing formation of the heart tube and causingthe concentric heart chamber phenotype. Presumptive atrial precursorcells are colored red, ventricular precursor cells are colored blue.Views are dorsal; anterior is up.

DETAILED DESCRIPTION

The invention provides methods of diagnosing, preventing, and treatingdiseases and conditions associated with PKCλ, such as diseases orconditions of the heart (also see below), and screening methods foridentifying compounds that can be used to treat or to prevent suchdiseases and conditions. In particular, we have identified a smallmolecule, concentramide, and a genetic mutation, heart-and-soul (has),which disrupt the earliest heart. Both cause the ventricle to formwithin the atrium. We show here that the has gene encodes an atypicalProtein Kinase C, Protein Kinase C λ (PKCλ). The has mutation results inthe disruption of epithelial cell-cell interactions in a broad range oftissues. Concentramide does not disrupt epithelial cell interactionsbut, rather, shifts the converging heart field of developing embryosrostrally. What is shared between the effects of concentramide and hasis a reversal of the order of fusion of the anterior and posterior endsof the heart field.

The diagnostic methods of the invention thus involve detection ofmutations in genes encoding PKCλ proteins, while the compoundidentification methods involve screening for compounds that affect thephenotype of organisms having mutations in genes encoding PKCλ or othermodels of appropriate diseases and conditions. The compoundidentification methods can also involve screening of candidate compoundsin the presence of concentramide, using organisms with or without a PKCλmutation (e.g., the has mutation). Compounds identified in this manner,as well as PKCλ genes and proteins themselves, can be used in methods totreat or prevent diseases and conditions associated with PKCλ.Compounds, antisense molecules, and antibodies that are found to inhibitPKCλ function can also be used to prevent or treat cancer.

The invention also provides animal model systems (e.g., zebrafish havingmutations (e.g., the heart and soul mutation) in PKCλ genes, or mice (orother animals) having such mutations) that can be used in the screeningmethods mentioned above, as well as the PKCλ protein, and genes encodingthis protein. Also included in the invention are genes encoding mutantzebrafish PKCλ proteins (e.g., genes having the heart and soul mutation)and proteins encoded by these genes. Antibodies that specifically bindto these proteins (wild type or mutant) are also included in theinvention.

The diagnostic, screening, and therapeutic methods of the invention, aswell as the animal model systems, proteins, and genes of the invention,are described further, as follows, after a brief description of diseasesand conditions associated with PKCλ, which can be diagnosed, prevented,or treated according to the invention.

PKCλ-Associated Diseases or Conditions

Abnormalities in PKCλ genes or proteins can be associated with any of awide variety of diseases or conditions, all of which can thus bediagnosed, prevented, or treated using the methods of the invention. Forexample, as discussed above, the heart and soul mutation in zebrafish ischaracterized by abnormal heart growth and development. Thus, detectionof abnormalities in PKCλ genes or their expression can be used inmethods to diagnose, or to monitor the treatment or development of,diseases or conditions of heart. In addition, compounds that areidentified in the screening methods described herein, as well as PKCλnucleic acid molecules, proteins, and antibodies themselves, can be usedin methods to prevent or treat such diseases or conditions.

Specific examples of diseases or conditions of the heart that can bediagnosed, prevented, or treated according to the invention includecongenital defects that result in heart malformation. These includecongenital defects, such as Ebstein anomaly, which results inabnormalities of the tricuspid valve, as well as isomerism defects,which are characterized by a wide variety of abnormalities in theasymmetrical arrangement of particular organs, such as the heart, organsof the digestive tract, and the spleen, that normally occurs duringdevelopment.

In right isomerism sequence, for example, which is also known asasplenia syndrome, Ivemark syndrome, and right atrial isomerism, theright side structures of the heart are duplicated on the left side ofthe heart, and the spleen is absent. This condition can lead to verycomplex and severe heart defects, such as atrioventricular septal defect(AVSD). In contrast, in left isomerism sequence, which is also known aspolysplenia syndrome, the left side heart structures are duplicated andmultiple small spleens may be present. This condition can lead to heartdefects as well, such as heart block, which results in a slow heartbeat, atrial septal defect, which is characterized by a hole between thetop two heart chambers, and AVSD. With both types of isomerisms,twisting of the bowel or intestinal obstruction may result, due to theincorrect positioning of the intestines. Related defects may occur inother organs, such as the kidney.

Other diseases and conditions related to PKCλ that can be diagnosed,prevented, or treated according to the invention include those that arecharacterized by abnormalities in tight junctions. As is noted above, wehave found that abnormalities in PKCλ (caused, e.g., by the hasmutation) can lead to defects in epithelial cell-cell interactions. Thisis due to abnormalities in the formation of tight junctions, which playcritical roles in the sealing of spaces between the individualepithelial or endothelial cells that make up sheets of these cells thatline the cavities of the body (e.g., the gastrointestinal tract, bloodvessels, the respiratory tract, and the urinary tract), as well asenclose and protect certain organs (e.g., the brain). These sheets ofcells function as selective permeability barriers, and alteration of thepermeability of these barriers, due to, e.g., a PKCλ defect, can lead toany of a number of diseases or conditions that are well known in theart. For example, increased permeability of the lining of thegastrointestinal tract can lead to Crohn's disease, acutegastroenteritis, and diarrhea. Also, defects in tight junctions caninterfere with the critical functions of the blood/brain barrier or theblood/retina barrier. As an additional example, vascular permeabilitydefects in diabetic patients can lead to conditions such as diabeticretinopathy. Additional diseases and conditions that can be diagnosed,prevented, or treated, according to the invention, include those thatare associated with abnormalities in epithelial cell polarity, such aspolycystic kidney disease (e.g., autosomal dominant polycystic kidneydisease). Also, because we have found that abnormalities in PKCλ lead todefects in cell growth control, a role for PKCλ in cancer is indicated.Compounds that are found to modulate PKCλ activity, thus, can be used inthe prevention and treatment of cancer, such as, for example, carcinomas(e.g., renal cell carcinoma), which are cancers derived from epithelialcells.

Diagnostic Methods

Nucleic acid molecules encoding PKCλ proteins, as well as polypeptidesencoded by these nucleic acid molecules and antibodies specific forthese polypeptides, can be used in methods to diagnose or to monitordiseases and conditions involving mutations in, or inappropriateexpression of, genes encoding this protein.

The diagnostic methods of the invention can be used, for example, withpatients that have a disease or condition associated with PKCλ, in aneffort to determine its etiology and, thus, to facilitate selection ofan appropriate course of treatment The diagnostic methods can also beused with patients who have not yet developed, but who are at risk ofdeveloping, such a disease or condition, or with patients that are at anearly stage of developing such a disease or condition. Also, thediagnostic methods of the invention can be used in prenatal geneticscreening, for example, to identify parents who may be carriers of arecessive mutation in a gene encoding a PKCλ protein. The methods of theinvention can be used to diagnose (or to treat) the disorders describedherein in any mammal, for example, in humans, domestic pets, orlivestock.

Abnormalities in PKCλ that can be detected using the diagnostic methodsof the invention include those characterized by, for example, (i) a geneencoding a PKCλ protein containing a mutation that results in theproduction of an abnormal PKCλ protein, (ii) an abnormal PKCλpolypeptide itself (e.g., a truncated protein), and (iii) a mutation ina PKCλ gene that results in production of an abnormal amount of thisprotein. Detection of such abnormalities can be used to diagnose humandiseases or conditions related to PKCλ, such as those affecting theheart. Exemplary of the mutations in PKCλ genes is the heart and soulmutation, which is described further below.

A mutation in a PKCλ gene can be detected in any tissue of a subject,even one in which this protein is not expressed. Because of the possiblylimited number of tissues in which these proteins may be expressed, forlimited time periods, and because of the possible undesirability ofsampling such tissues (e.g., heart tissue) for assays, it may bepreferable to detect mutant genes in other, more easily obtained sampletypes, such as in blood or amniotic fluid samples.

Detection of a mutation in a gene encoding a PKCλ protein can be carriedout using any standard diagnostic technique. For example, a biologicalsample obtained from a patient can be analyzed for one or more mutations(e.g., a heart and soul mutation) in nucleic acid molecules encoding aPKCλ protein using a mismatch detection approach. Generally, thisapproach involves polyrnerase chain reaction (PCR) amplification ofnucleic acid molecules from a patient sample, followed by identificationof a mutation (i.e., a mismatch) by detection of altered hybridization,aberrant electrophoretic gel migration, binding, or cleavage mediated bymismatch binding proteins, or by direct nucleic acid moleculesequencing. Any of these techniques can be used to facilitate detectionof a mutant gene encoding a PKCλ protein, and each is well known in theart. For instance, examples of these techniques are described by Oritaet al. (Proc. Natl. Acad. Sci. U.S.A. 86:2766-2770, 1989) and Sheffieldet al. (Proc. Natl. Acad. Sci. U.S.A. 86:232-236, 1989).

As noted above, in addition to facilitating diagnosis of an existingdisease or condition, mutation detection assays also provide anopportunity to diagnose a predisposition to disease related to amutation in a PKCλ gene before the onset of symptoms. For example, apatient who is heterozygous for a gene encoding an abnormal PKCλ protein(or an abnormal amount thereof) that suppresses normal PKCλ biologicalactivity or expression may show no clinical symptoms of a diseaserelated to such proteins, and yet possess a higher than normalprobability of developing such disease. Given such a diagnosis, apatient can take precautions to minimize exposure to adverseenvironmental factors, and can carefully monitor their medicalcondition, for example, through frequent physical examinations. Asmentioned above, this type of diagnostic approach can also be used todetect a mutation in a gene encoding the PKCλ protein in prenatalscreens.

While it may be preferable to carry out diagnostic methods for detectinga mutation in a PKCλ gene using genomic DNA from readily accessibletissues, as noted above, mRNA encoding this protein, or the proteinitself, can also be assayed from tissue samples in which it isexpressed. Expression levels of a gene encoding PKCλ in such a tissuesample from a patient can be determined by using any of a number ofstandard techniques that are well known in the art, including northernblot analysis and quantitative PCR (see, e.g., Ausubel et al., supra;PCR Technology: Principles and Applications for DNA Amplification, H. A.Ehrlich, Ed., Stockton Press, NY; Yap et al. Nucl. Acids. Res. 19:4294,1991).

In another diagnostic approach of the invention, an immunoassay is usedto detect or to monitor the level of a PKCλ protein in a biologicalsample. Polyclonal or monoclonal antibodies specific for the PKCλprotein can be used in any standard immunoassay format (e.g., ELISA,Western blot, or RIA; see, e.g., Ausubel et al., supra) to measurepolypeptide the levels of PKCλ. These levels can be compared to levelsof PKCλ in a sample from an unaffected individual. Detection of adecrease in production of PKCλ using this method, for example, may beindicative of a condition or a predisposition to a condition involvinginsufficient biological activity of the PKCλ protein.

Imnunohistochemical techniques can also be utilized for detection ofPKCλ protein in patient samples. For example, a tissue sample can beobtained from a patient, sectioned, and stained for the presence of PKCλusing an anti-PKCλ antibody and any standard detection system (e.g., onethat includes a secondary antibody conjugated to an enzyme, such ashorseradish peroxidase). General guidance regarding such techniques canbe found in, e.g., Bancroft et al., Theory and Practice of HistologicalTechniques, Churchill Livingstone, 1982, and Ausubel et al., supra.

Identification of Molecules that can be Used to Treat or to PreventDiseases or Conditions Associated with PKCλ

Identification of a mutation in the gene encoding PKCλ as resulting in aphenotype that results in abnormal heart growth and developmentfacilitates the identification of molecules (e.g., small organic orinorganic molecules, antibodies, peptides, or nucleic acid molecules)that can be used to treat or to prevent diseases or conditionsassociated with PKCλ, as discussed above. The effects of candidatecompounds on such diseases or conditions can be investigated using, forexample, the zebrafish system. As is mentioned above, the zebrafish,Danio rerio, is a convenient organism to use in the genetic analysis ofdevelopment. It has an accessible and transparent embryo, allowingdirect observation of organ function from the earliest stages ofdevelopment, has a short generation time, and is fecund. As discussedfurther below, zebrafish and other animals having a PKCλ mutation, suchas the heart and soul mutation, which can be used in these methods, arealso included in the invention.

In one example of the screening methods of the invention, a zebrafishhaving a mutation in a gene encoding the PKCλ protein (e.g., a zebrafishhaving the heart and soul mutation) is contacted with a candidatecompound, and the effect of the compound on the development of a heartabnormality, or on the status of such an existing abnormality, ismonitored relative to an untreated, identically mutant control. In avariation of this method, a zebrafish, with or without a mutation in thePKCλ gene (e.g., the has mutation), is contacted with a candidatecompound in the presence of concentramide.

After a compound has been shown to have a desired effect in thezebrafish system, it can be tested in other models of heart disease, forexample, in mice or other animals having a mutation in a gene encodingPKCλ. Alternatively, testing in such animal model systems can be carriedout in the absence of zebrafish testing. Compounds of the invention canalso be tested in animal models of cancer.

Cell culture-based assays can also be used in the identification ofmolecules that increase or decrease PKCλ levels or biological activity.According to one approach, candidate molecules are added at varyingconcentrations to the culture medium of cells expressing PKCλ mRNA. PKCλbiological activity is then measured using standard techniques. Themeasurement of biological activity can include the measurement of PKCλprotein and nucleic acid molecule levels.

In general, novel drugs for the prevention or treatment of diseasesrelated to mutations in genes encoding PKCλ can be identified from largelibraries of natural products, synthetic (or semi-synthetic) extracts,and chemical libraries using methods that are well known in the art.Those skilled in the field of drug discovery and development willunderstand that the precise source of test extracts or compounds is notcritical to the screening methods of the invention and thatdereplication, or the elimination of replicates or repeats of materialsalready known for their therapeutic activities for PKCλ, can be employedwhenever possible.

Candidate compounds to be tested include purified (or substantiallypurified) molecules or one or more components of a mixture of compounds(e.g., an extract or supernatant obtained from cells; Ausubel et al.,supra), and such compounds further include both naturally occurring orartificially derived chemicals and modifications of existing compounds.For example, candidate compounds can be polypeptides, synthesizedorganic or inorganic molecules, naturally occurring organic or inorganicmolecules, nucleic acid molecules, and components thereof.

Numerous sources of naturally occurring candidate compounds are readilyavailable to those skilled in the art. For example, naturally occurringcompounds can be found in cell (including plant, fungal, prokaryotic,and animal) extracts, mammalian serum, growth medium in which mammaliancells have been cultured, protein expression libraries, or fermentationbroths. In addition, libraries of natural compounds in the form ofbacterial, fungal, plant, and animal extracts are commercially availablefrom a number of sources, including Biotics (Sussex, UK), Xenova(Slough, UK), Harbor Branch Oceanographic Institute (Ft. Pierce, FL),and PharmaMar, U.S.A. (Cambridge, Mass.). Furthermore, libraries ofnatural compounds can be produced, if desired, according to methods thatare known in the art, e.g., by standard extraction and fractionation.

Artificially derived candidate compounds are also readily available tothose skilled in the art. Numerous methods are available for generatingrandom or directed synthesis (e.g., semi-synthesis or total synthesis)of any number of chemical compounds, including, for example,saccharide-, lipid-, peptide-, and nucleic acid molecule-basedcompounds. In addition, synthetic compound libraries are commerciallyavailable from Brandon Associates (Merrimack, N.H.) and AldrichChemicals (Milwaukee, Wis.). Libraries of synthetic compounds can alsobe produced, if desired, according to methods known in the art, e.g., bystandard extraction and fractionation. Furthermore, if desired, anylibrary or compound can be readily modified using standard chemical,physical, or biochemical methods. The techniques of modem syntheticchemistry, including combinatorial chemistry, can also be used (reviewedin Schreiber, Bioorganic and Medicinal Chemistry 6:1172-1152, 1998;Schreiber, Science 287:1964-1969, 2000).

When a crude extract is found to have an effect on the development orpersistence of a PKCλ-associated disease, further fractionation of thepositive lead extract can be carried out to isolate chemicalconstituents responsible for the observed effect. Thus, the goal of theextraction, fractionation, and purification process is the carefulcharacterization and identification of a chemical entity within thecrude extract having a desired activity. The same assays describedherein for the detection of activities in mixtures of compounds can beused to purify the active component and to test derivatives of thesecompounds. Methods of fractionation and purification of suchheterogeneous extracts are well known in the art. If desired, compoundsshown to be useful agents for treatment can be chemically modifiedaccording to methods known in the art.

In general, compounds that are found to activate PKCλ expression oractivity may be used in the prevention or treatment of diseases orconditions of heart, such as those that are characterized by abnormalgrowth or development, or heart failure (also see above). Compounds thatare found to modulate, e.g., block PKCλ expression or activity may beused to prevent or to treat cancer.

Animal Model Systems

The invention also provides animal model systems for use in carrying outthe screening methods described above. Examples of these model systemsinclude zebrafish and other animals, such as mice, that have a mutation(e.g., the heart and soul mutation) in a PKCλ gene. For example, azebrafish model that can be used in the invention can include a mutationthat results in a lack of PKCλ protein production or production of atruncated (e.g., by introduction of a stop codon) or otherwise alteredPKCλ gene product. As a specific example, a zebrafish having the heartand soul mutation can be used (see below).

Treatment or Prevention of PKCλ-Associated Diseases or Conditions

Compounds identified using the screening methods described above can beused to treat patients that have or are at risk of developing diseasesor conditions of the heart or cancer. Nucleic acid molecules encodingthe PKCλ protein, as well as these proteins themselves, can also be usedin such methods. Treatment may be required only for a short period oftime or may, in some form, be required throughout a patient's lifetime.Any continued need for treatment, however, can be determined using, forexample, the diagnostic methods described above. In considering varioustherapies, it is to be understood that such therapies are, preferably,targeted to the affected or potentially affected organ (e.g., theheart). Such targeting can be achieved using standard methods.

Treatment or prevention of diseases resulting from a mutated PKCλ genecan be accomplished, for example, by modulating the function of a mutantPKCλ protein. Treatment can also be accomplished by delivering normalPKCλ protein to appropriate cells, altering the levels of normal ormutant PKCλ protein, replacing a mutant gene encoding a PKCλ proteinwith a normal gene encoding a PKCλ protein, or administering a normalgene encoding a PKCλ protein. It is also possible to correct the effectsof a defect in a gene encoding a PKCλ protein by modifying thephysiological pathway (e.g., a signal transduction pathway) in which aPKCλ protein participates. in a patient diagnosed as being heterozygousfor a gene encoding a mutant PKCλ protein, or as susceptible to suchmutations or aberrant PKCλ expression (even if those mutations orexpression patterns do not yet result in alterations in expression orbiological activity of PKCλ), any of the therapies described herein canbe administered before the occurrence of the disease phenotype. Inparticular, compounds shown to have an effect on the phenotype ofmutants, or to modulate expression of PKCλ proteins, can be administeredto patients diagnosed with potential or actual disease by any standarddosage and route of administration.

Any appropriate route of administration can be employed to administer acompound identified as described above, a PKCλ gene, or a PKCλ protein,according to the invention. For example, administration can beparenteral, intravenous, intra-arterial, subcutaneous, intramuscular,intraventricular, intracapsular, intraspinal, intracistemal,intraperitoneal, intranasal, by aerosol, by suppository, or oral.

A therapeutic compound of the invention can be administered within apharmaceutically acceptable diluent, carrier, or excipient, in unitdosage form. Administration can begin before or after the patient issymptomatic. Methods that are well known in the art for makingformulations are found, for example, in Remington's PharmaceuticalSciences (18^(th) edition), ed. A. Gennaro, 1990, Mack PublishingCompany, Easton, Pa. Therapeutic formulations can be in the form ofliquid solutions or suspensions. Formulations for parenteraladministration can contain, for example, excipients, sterile water, orsaline; polyalkylene glycols, such as polyethylene glycol; oils ofvegetable origin; or hydrogenated napthalenes. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers can be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. For oraladministration, formulations can be in the form of tablets or capsules.Formulations for inhalation can contain excipients, for example,lactose, or can be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholate, or canbe oily solutions for administration in the form of nasal drops or as agel. Alternatively, intranasal formulations can be in the form ofpowders or aerosols.

To replace a mutant protein with normal protein, or to add protein tocells that do not express a sufficient amount of PKCλ or normal PKCλ, itmay be necessary to obtain large amounts of pure PKCλ protein from cellculture systems in which the protein is expressed (see, e.g., below).Delivery of the protein to the affected tissue can then be accomplishedusing appropriate packaging or administration systems.

Gene therapy is another therapeutic approach for preventing orameliorating diseases caused by PKCλ gene defects. Nucleic acidmolecules encoding wild type PKCλ protein can be delivered to cells thatlack sufficient, normal PKCλ protein biological activity (e.g., cellscarrying mutations (e.g., the heart and soul mutation) in PKCλ genes).The nucleic acid molecules must be delivered to those cells in a form inwhich they can be taken up by the cells and so that sufficient levels ofprotein, to provide effective PKCλ protein function, can be produced.Alternatively, for some PKCλ mutations, it may be possible to slow theprogression of the resulting disease or to modulate PKCλ proteinactivity by introducing another copy of a homologous gene bearing asecond mutation in that gene, to alter the mutation, or to use anothergene to block any negative effect.

Transducing viral (e.g., retroviral, adenoviral, and adeno-associatedviral) vectors can be used for somatic cell gene therapy, especiallybecause of their high efficiency of infection and stable integration andexpression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430,1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer etal., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A.94:10319, 1997). For example, the full length PKCλ gene, or a portionthereof, can be cloned into a retroviral vector and expression can bedriven from its endogenous promoter, from the retroviral long terminalrepeat, or from a promoter specific for a target cell type of interest.Other viral vectors that can be used include, for example, a vacciniavirus, a bovine papilloma virus, or a herpes virus, such as Epstein-BarrVirus (also see, for example, the vectors of Miller, Human Gene Therapy15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al.,BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion inBiotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991;Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322,1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416,1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle etal., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995).Retroviral vectors are particularly well developed and have been used inclinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990;Anderson et al., U.S. Pat. No. 5,399,346).

Non-viral approaches can also be employed for the introduction oftherapeutic DNA into cells predicted to be subject to diseases involvingthe PKCλ protein. For example, a PKCλ nucleic acid molecule or anantisense nucleic acid molecule can be introduced into a cell bylipofection (Felgner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413,1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am.J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al.,Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal ofBiological Chemistry 264:16985, 1989), or by micro-injection undersurgical conditions (Wolff et al., Science 247:1465, 1990).

Gene transfer can also be achieved using non-viral means involvingtransfection in vitro. Such methods include the use of calciumphosphate, DEAE dextran, electroporation, and protoplast fusion.Liposomes can also be potentially beneficial for delivery of DNA into acell. Transplantation of normal genes into the affected tissues of apatient can also be accomplished by transferring a normal PKCλ proteininto a cultivatable cell type ex vivo (e.g., an autologous orheterologous primary cell or progeny thereof), after which the cell (orits descendants) are injected into a targeted tissue.

PKCλ cDNA expression for use in gene therapy methods can be directedfrom any suitable promoter (e.g., the human cytomegalovirus (CMV),simian virus 40 (SV40), or metallothionein promoters), and regulated byany appropriate mammalian regulatory element. For example, if desired,enhancers known to preferentially direct gene expression in specificcell types can be used to direct PKCλ expression. The enhancers used caninclude, without limitation, those that are characterized as tissue- orcell-specific enhancers. Alternatively, if a PKCλ genomic clone is usedas a therapeutic construct (such clones can be identified byhybridization with PKCλ cDNA, as described herein), regulation can bemediated by the cognate regulatory sequences or, if desired, byregulatory sequences derived from a heterologous source, including anyof the promoters or regulatory elements described above.

Molecules for effecting antisense-based strategies can be employed toexplore PKCλ protein gene function, as a basis for therapeutic drugdesign, as well as to treat PKCλ-associated diseases, such as cancer.These strategies are based on the principle that sequence-specificsuppression of gene expression (via transcription or translation) can beachieved by intracellular hybridization between genomic DNA or MRNA anda complementary antisense species. The formation of a hybrid RNA duplexinterferes with transcription of the target PKCλ-encoding genomic DNAmolecule, or processing, transport, translation, or stability of thetarget PKCλ mRNA molecule.

Antisense strategies can be delivered by a variety of approaches. Forexample, antisense oligonucleotides or antisense RNA can be directlyadministered (e.g., by intravenous injection) to a subject in a formthat allows uptake into cells. Alternatively, viral or plasmid vectorsthat encode antisense RNA (or antisense RNA fragments) can be introducedinto a cell in vivo or ex vivo. Antisense effects can be induced bycontrol (sense) sequences; however, the extent of phenotypic changes ishighly variable. Phenotypic effects induced by antisense molecules arebased on changes in criteria such as protein levels, protein activitymeasurement, and target mRNA levels.

PKCλ gene therapy can also be accomplished by direct administration ofantisense PKCλ MRNA to a cell that is expected to be adversely affectedby the expression of wild type or mutant PKCλ protein. The antisensePKCλ mRNA can be produced and isolated by any standard technique, but ismost readily produced by in vitro transcription using an antisense PKCλcDNA under the control of a high efficiency promoter (e.g., the T7promoter). Administration of antisense PKCλ MRNA to cells can be carriedout by any of the methods for direct nucleic acid moleculeadministration described above.

An alternative strategy for inhibiting PKCλ protein function using genetherapy involves intracellular expression of an anti-PKCλ proteinantibody or a portion of an anti-PKCλ protein antibody. For example, thegene (or gene fragment) encoding a monoclonal antibody that specificallybinds to a PKCλ protein and inhibits its biological activity can beplaced under the transcriptional control of a tissue-specific generegulatory sequence.

Another therapeutic approach included in the invention involvesadministration of a recombinant PKCλ polypeptide, either directly to thesite of a potential or actual disease-affected tissue (for example, byinjection) or systemically (for example, by any conventional recombinantprotein administration technique). The dosage of the PKCλ proteindepends on a number of factors, including the size and health of theindividual patient but, generally, between 0.1 mg and 100 mg, inclusive,is administered per day to an adult in any pharmaceutically acceptableformulation.

In addition to the therapeutic methods described herein, involvingadministration of PKCλ-modulating compounds, PKCλ proteins, or PKCλnucleic acids to patients, the invention provides methods of culturingorgans in the presence of such molecules. In particular, as is notedabove, a PKCλ mutation is associated with abnormal heart growth anddevelopment. Thus, culturing heart tissue in the presence of thesemolecules can be used to promote its growth and development. This tissuecan be that which is being prepared for transplant from, e.g., anallogeneic or xenogeneic donor, as well as synthetic tissue or organs.

Synthesis of PKCλ Proteins, Polypeptides, and Polypeptide Fragments

Those skilled in the art of molecular biology will understand that awide variety of expression systems can be used to produce recombinantPKCλ proteins. As discussed further below, the precise host cell used isnot critical to the invention. The PKCλ proteins can be produced in aprokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., S.cerevisiae, insect cells, such as Sf9 cells, or mammalian cells, such asCOS-1, NIH 3T3, or HeLa cells). These cells are commercially availablefrom, for example, the American Type Culture Collection, Manassas, Va.(see also Ausubel et al., supra). The method of transformation and thechoice of expression vehicle (e.g., expression vector) will depend onthe host system selected. Transformation and transfection methods aredescribed, e.g., in Ausubel et al., supra, and expression vehicles canbe chosen from those provided, e.g., in Pouwels et al., Cloning Vectors:A Laboratory Manual, 1985, Supp. 1987. Specific examples of expressionsystems that can be used in the invention are described further asfollows.

For protein expression, eukaryotic or prokaryotic expression systems canbe generated in which PKCλ gene sequences are introduced into a plasmidor other vector, which is then used to transform living cells.Constructs in which full-length PKCλ cDNAs, containing the entire openreading frame, inserted in the correct orientation into an expressionplasmid, can be used for protein expression. Alternatively, portions ofPKCλ gene sequences, including wild type or mutant PKCλ sequences, canbe inserted. Prokaryotic and eukaryotic expression systems allow variousimportant functional domains of PKCλ proteins to be recovered, ifdesired, as fusion proteins, and then used for binding, structural, andfunctional studies, and also for the generation of antibodies.

Typical expression vectors contain promoters that direct synthesis oflarge amounts of mRNA corresponding to a nucleic acid molecule that hasbeen inserted into the vector. They can also include a eukaryotic orprokaryotic origin of replication, allowing for autonomous replicationwithin a host cell, sequences that confer resistance to an otherwisetoxic drug, thus allowing vector-containing cells to be selected in thepresence of the drug, and sequences that increase the efficiency withwhich the synthesized mRNA is translated. Stable, long-term vectors canbe maintained as freely replicating entities by using regulatoryelements of, for example, viruses (e.g., the OriP sequences from theEpstein Barr Virus genome). Cell lines can also be produced that havethe vector integrated into genomic DNA of the cells and, in this manner,the gene product can be produced in the cells on a continuous basis.

Expression of foreign molecules in bacteria, such as Escherichia coli,requires the insertion of a foreign nucleic acid molecule, e.g., a PKCλnucleic acid molecule, into a bacterial expression vector. Such plasmidvectors include several elements required for the propagation of theplasmid in bacteria, and for expression of foreign DNA contained withinthe plasmid. Propagation of only plasmid-bearing bacteria is achieved byintroducing, into the plasmid, a selectable marker-encoding gene thatallows plasmid-bearing bacteria to grow in the presence of an otherwisetoxic drug. The plasmid also contains a transcriptional promoter capableof directing synthesis of large amounts of mRNA from the foreign DNA.Such promoters can be, but are not necessarily, inducible promoters thatinitiate transcription upon induction by culture under appropriateconditions (e.g., in the presence of a drug that activates thepromoter). The plasmid also, preferably, contains a polylinker tosimplify insertion of the gene in the correct orientation within thevector.

Once an appropriate expression vector containing a PKCλ gene, or afragment, fusion, or mutant thereof, is constructed, it can beintroduced into an appropriate host cell using a transformationtechnique, such as, for example, calcium phosphate transfection,DEAE-dextran transfection, electroporation, microinjection, protoplastfusion, or liposome-mediated transfection. Host cells that can betransfected with the vectors of the invention can include, but are notlimited to, E. coli or other bacteria, yeast, fungi, insect cells(using, for example, baculoviral vectors for expression), or cellsderived from mice, humans, or other animals. Mammalian cells can also beused to express PKCλ proteins using a virus expression system (e.g., avaccinia virus expression system) described, for example, in Ausubel etal., supra.

In vitro expression of PKCλ proteins, fusions, polypeptide fragments, ormutants encoded by cloned DNA can also be carried out using the T7late-promoter expression system. This system depends on the regulatedexpression of T7 RNA polymerase, an enzyme encoded in the DNA ofbacteriophage T7. The T7 RNA polymerase initiates transcription at aspecific 23 base pair promoter sequence called the T7 late promoter.Copies of the T7 late promoter are located at several sites on the T7genome, but none are present in E. coli chromosomal DNA. As a result, inT7-infected E. coli, T7 RNA polymerase catalyzes transcription of viralgenes, but not E. coli genes. In this expression system, recombinant E.coli cells are first engineered to carry the gene encoding T7 RNApolymerase next to the lac promoter. In the presence of IPTG, thesecells transcribe the T7 polymerase gene at a high rate and synthesizeabundant amounts of T7 RNA polymerase. These cells are then transformedwith plasmid vectors that carry a copy of the T7 late promoter protein.When IPTG is added to the culture medium containing these transformed E.coli cells, large amounts of T7 RNA polymerase are produced. Thepolymerase then binds to the T7 late promoter on the plasmid expressionvectors, catalyzing transcription of the inserted cDNA at a high rate.Since each E. coli cell contains many copies of the expression vector,large amounts of mRNA corresponding to the cloned cDNA can be producedin this system and the resulting protein can be radioactively labeled.

Plasmid vectors containing late promoters and the corresponding RNApolymerases from related bacteriophages, such as T3, T5, and SP6, canalso be used for in vitro production of proteins from cloned DNA. E.coli can also be used for expression using an M13 phage, such as mGPI-2.Furthermore, vectors that contain phage lambda regulatory sequences, orvectors that direct the expression of fusion proteins, for example, amaltose-binding protein fusion protein or a glutathione-S-transferasefusion protein, also can be used for expression in E. coli.

Eukaryotic expression systems are useful for obtaining appropriatepost-translational modification of expressed proteins. Transienttransfection of a eukaryotic expression plasmid containing a PKCλ geneinto a eukaryotic host cell allows the transient production of a PKCλprotein by the transfected host cell. PKCλ proteins can also be producedby a stably-transfected eukaryotic (e.g., mammalian) cell line. A numberof vectors suitable for stable transfection of mammalian cells areavailable to the public (see, e.g., Pouwels et al., supra), as aremethods for constructing lines including such cells (see, e.g., Ausubelet al., supra).

In one example, cDNA encoding a PKCλ protein, fusion, mutant, orpolypeptide fragment is cloned into an expression vector that includesthe dihydrofolate reductase (DHFR) gene. Integration of the plasmid and,therefore, integration of the heart and soul protein-encoding gene, intothe host cell chromosome is selected for by inclusion of 0.01-300 μMmethotrexate in the cell culture medium (Ausubel et al., supra). Thisdominant selection can be accomplished in most cell types. Recombinantprotein expression can be increased by DHFR-mediated amplification ofthe transfected gene. Methods for selecting cell lines bearing geneamplifications are described in Ausubel et al., supra. These methodsgenerally involve extended culture in medium containing graduallyincreasing levels of methotrexate. The most commonly usedDHFR-containing expression vectors are pCVSEII-DHFR and pAdD26SV(A)(described, for example, in Ausubel et al., supra). The host cellsdescribed above or, preferably, a DHFR-deficient CHO cell line (e.g.,CHO DHFR-cells, ATCC Accession No. CRL 9096) are among those that aremost preferred for DHFR selection of a stably transfected cell line orDHFR-mediated gene amplification.

Another preferred eukaryotic expression system is the baculovirus systemusing, for example, the vector pBacPAK9, which is available fromClontech (Palo Alto, Calif.). If desired, this system can be used inconjunction with other protein expression techniques, for example, themyc tag approach described by Evan et al. (Molecular and CellularBiology 5:3610-3616, 1985).

Once a recombinant protein is expressed, it can be isolated from theexpressing cells by cell lysis followed by protein purificationtechniques, such as affinity chromatography. In this example, ananti-PKCλ antibody, which can be produced by the methods describedherein, can be attached to a column and used to isolate the recombinantPKCλ. Lysis and fractionation of PKCλ-harboring cells prior to affinitychromatography can be performed by standard methods (see, e.g., Ausubelet al., supra). Once isolated, the recombinant protein can, if desired,be purified further by, e.g., high performance liquid chromatography(HPLC; e.g., see Fisher, Laboratory Techniques In Biochemistry andMolecular Biology, Work and Burdon, Eds., Elsevier, 1980).

Polypeptides of the invention, particularly short PKCλ fragments andlonger fragments of the N-terminus and C-terminus of PKCλ, can also beproduced by chemical synthesis (e.g., by the methods described in SolidPhase Peptide Synthesis, 2^(nd) ed., 1984, The Pierce Chemical Co.,Rockford, Ill.). These general techniques of polypeptide expression andpurification can also be used to produce and isolate useful PKCλfragments or analogs, as described herein.

PKCλ Protein Fragments

Polypeptide fragments that include various portions of PKCλ proteins areuseful in identifying the domains of PKCλ that are important for itsbiological activities. Methods for generating such fragments are wellknown in the art (see, for example, Ausubel et al., supra), using thenucleotide sequences provided herein. For example, a PKCλ proteinfragment can be generated by PCR amplifying a desired PKCλ nucleic acidmolecule fragment using oligonucleotide primers designed based upon PKCλnucleic acid sequences. Preferably, the oligonucleotide primers includeunique restriction enzyme sites that facilitate insertion of theamplified fragment into the cloning site of an expression vector (e.g.,a mammalian expression vector, see above). This vector can then beintroduced into a cell (e.g., a mammalian cell; see above) by artifice,using any of the various techniques that are known in the art, such asthose described herein, resulting in the production of a PKCλ proteinfragment in the cell containing the expression vector. PKCλ proteinfragments (e.g., chimeric fusion proteins) can also be used to raiseantibodies specific for various regions of the PKCλ protein using, forexample, the methods described below.

PKCλ Protein Antibodies

To prepare polyclonal antibodies, PKCλ proteins, fragments of PKCλproteins, or fusion proteins containing defined portions of PKCλproteins can be synthesized in, e.g., bacteria by expression ofcorresponding DNA sequences contained in a suitable cloning vehicle.Fusion proteins are commonly used as a source of antigen for producingantibodies. Two widely used expression systems for E. coli are lacZfusions using the pUR series of vectors and trpE fusions using the pATHvectors. The proteins can be purified, coupled to a carrier protein,mixed with Freund's adjuvant to enhance stimulation of the antigenicresponse in an inoculated animal, and injected into rabbits or otherlaboratory animals. Alternatively, protein can be isolated fromPKCλ-expressing cultured cells. Following booster injections atbi-weekly intervals, the rabbits or other laboratory animals are thenbled and the sera isolated. The sera can be used directly or can bepurified prior to use by various methods, including affinitychromatography employing reagents such as Protein A-Sepharose,antigen-Sepharose, and anti-mouse-Ig-Sepharose. The sera can then beused to probe protein extracts from PKCλ-expressing tissue fractionatedby polyacrylamide gel electrophoresis to identify PKCλ proteins.Alternatively, synthetic peptides can be made that correspond toantigenic portions of the protein and used to inoculate the animals.

To generate peptide or full-length protein for use in making, forexample, PKCλ-specific antibodies, a PKCλ coding sequence can beexpressed as a C-terminal or N-terminal fusion with glutathioneS-transferase (GST; Smith et al., Gene 67:31-40, 1988). The fusionprotein can be purified on glutathione-Sepharose beads, eluted withglutathione, cleaved with a protease, such as thrombin or Factor-Xa (atthe engineered cleavage site), and purified to the degree required tosuccessfully immunize rabbits. Primary immunizations can be carried outwith Freund's complete adjuvant and subsequent immunizations performedwith Freund's incomplete adjuvant. Antibody titers can be monitored byWestern blot and immunoprecipitation analyses using the protease-cleavedPKCλ fragment of the GST-PKCλ protein. Immune sera can be affinitypurified using CNBr-Sepharose-coupled PKCλ. Antiserum specificity can bedetermined using a panel of unrelated GST fusion proteins.

Alternatively, monoclonal PKCλ antibodies can be produced by using, asan antigen, PKCλ isolated from PKCλ-expressing cultured cells or PKCλprotein isolated from tissues. The cell extracts, or recombinant proteinextracts containing PKCλ, can, for example, be injected with Freund'sadjuvant into mice. Several days after being injected, the mouse spleenscan be removed, the tissues disaggregated, and the spleen cellssuspended in phosphate buffered saline (PBS). The spleen cells serve asa source of lymphocytes, some of which would be producing antibody ofthe appropriate specificity. These can then be fused with permanentlygrowing myeloma partner cells, and the products of the fusion platedinto a number of tissue culture wells in the presence of selectiveagents, such as hypoxanthine, aminopterine, and thymidine (HAT). Thewells can then be screened by ELISA to identify those containing cellsmaking antibodies capable of binding to PKCλ, polypeptide fragment, ormutant thereof. These cells can then be re-plated and, after a period ofgrowth, the wells containing these cells can be screened again toidentify antibody-producing cells. Several cloning procedures can becarried out until over 90% of the wells contain single clones that arepositive for specific antibody production. From this procedure, a stableline of clones that produce the antibody can be established. Themonoclonal antibody can then be purified by affinity chromatographyusing Protein A Sepharose and ion exchange chromatography, as well asvariations and combinations of these techniques. Once produced,monoclonal antibodies are also tested for specific PKCλ recognition byWestern blot or immunoprecipitation analysis (see, e.g., Kohler et al.,Nature 256:495, 1975; Kohler et al., European Journal of Immunology6:511, 1976; Kohler et al., European Journal of Immunology 6:292, 1976;Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas,Elsevier, New York, N.Y., 1981; Ausubel et al., supra).

As an alternate or adjunct immunogen to GST fusion proteins, peptidescorresponding to relatively unique hydrophilic regions of PKCλ can begenerated and coupled to keyhole limpet hemocyanin (KLH) through anintroduced C-terminal lysine. Antiserum to each of these peptides can besimilarly affinity-purified on peptides conjugated to BSA, andspecificity tested by ELISA and Western blotting using peptideconjugates, and by Western blotting and immunoprecipitation using PKCλ,for example, expressed as a GST fusion protein.

Antibodies of the invention can be produced using PKCλ amino acidsequences that do not reside within highly conserved regions, and thatappear likely to be antigenic, as analyzed by criteria such as thoseprovided by the Peptide Structure Program (Genetics Computer GroupSequence Analysis Package, Program Manual for the GCG Package, Version7, 1991) using the algorithm of Jameson et al., CABIOS 4:181, 1988.These fragments can be generated by standard techniques, e.g., by PCR,and cloned into the pGEX expression vector. GST fusion proteins can beexpressed in E. coli and purified using a glutathione-agarose affinitymatrix (Ausubel et al., supra). To generate rabbit polyclonalantibodies, and to minimize the potential for obtaining antisera that isnon-specific, or exhibits low-affinity binding to PKCλ, two or threefusions are generated for each protein, and each fusion is injected intoat least two rabbits. Antisera are raised by injections in series,preferably including at least three booster injections.

In addition to intact monoclonal and polyclonal anti-PKCλ antibodies,the invention features various genetically engineered antibodies,humanized antibodies, and antibody fragments, including F(ab′)2, Fab′,Fab, Fv, and sFv fragments. Truncated versions of monoclonal antibodies,for example, can be produced by recombinant methods in which plasmidsare generated that express the desired monoclonal antibody fragment(s)in a suitable host. Antibodies can be humanized by methods known in theart, e.g., monoclonal antibodies with a desired binding specificity canbe commercially humanized (Scotgene, Scotland; Oxford Molecular, PaloAlto, Calif.). Fully human antibodies, such as those expressed intransgenic animals, are also included in the invention (Green et al.,Nature Genetics 7:13-21, 1994).

Ladner (U.S. Pat. Nos. 4,946,778 and 4,704,692) describes methods forpreparing single polypeptide chain antibodies. Ward et al., Nature341:544-546, 1989, describes the preparation of heavy chain variabledomains, which they term “single domain antibodies,” and which have highantigen-binding affinities. McCafferty et al., Nature 348:552-554, 1990,shows that complete antibody V domains can be displayed on the surfaceof fd bacteriophage, that the phage bind specifically to antigen, andthat rare phage (one in a million) can be isolated after affinitychromatography. Boss et al., U.S. Pat. No. 4,816,397, describes variousmethods for producing immunoglobulins, and immunologically functionalfragments thereof, that include at least the variable domains of theheavy and light chains in a single host cell. Cabilly et al., U.S. Pat.No. 4,816,567, describes methods for preparing chimeric antibodies.

Use of PKCλ Antibodies

Antibodies to PKCλ can be used, as noted above, to detect PKCλ or toinhibit the biological activities of PKCλ. For example, a nucleic acidmolecule encoding an antibody or portion of an antibody can be expressedwithin a cell to inhibit PKCλ function. In addition, the antibodies canbe coupled to compounds, such as radionuclides and liposomes, fordiagnostic or therapeutic uses. Antibodies that inhibit the activity ofa PKCλ polypeptide described herein can also be useful in preventing orslowing the development of a disease caused by inappropriate expressionof a wild type or mutant PKCλ gene.

Detection of PKCλ Gene Expression

As noted, the antibodies described above can be used to monitor PKCλgene expression. In situ hybridization of RNA can be used to detect theexpression of PKCλ genes. RNA in situ hybridization techniques rely uponthe hybridization of a specifically labeled nucleic acid probe to thecellular RNA in individual cells or tissues. Therefore, RNA in situhybridization is a powerful approach for studying tissue- andtemporal-specific gene expression. In this method, oligonucleotides,cloned DNA fragments, or antisense RNA transcripts of cloned DNAfragments corresponding to unique portions of PKCλ genes are used todetect specific mRNA species, e.g., in the tissues of animals, such asmice, at various developmental stages. Other gene expression detectiontechniques are known to those of skill in the art and can be employedfor detection of PKCλ gene expression.

Identification of Additional PKCλ Genes

Standard techniques, such as the polymerase chain reaction (PCR) and DNAhybridization, can be used to clone PKCλ gene homologues in otherspecies and PKCλ-related genes in humans. PKCλ-related genes andhomologues can be readily identified using low-stringency DNAhybridization or low-stringency PCR with human PKCλ probes or primers.Degenerate primers encoding human PKCλ or human PKCλ-related amino acidsequences can be used to clone additional PKCλ-related genes andhomologues by RT-PCR.

Construction of Transgenic Animals and Knockout Animals

Characterization of PKCλ genes provides information that allows PKCλknockout animal models to be developed by homologous recombination.Preferably, a PKCλ knockout animal is a mammal, most preferably a mouse.Similarly, animal models of PKCλ overproduction can be generated byintegrating one or more PKCλ sequences into the genome of an animal,according to standard transgenic techniques. Moreover, the effect ofPKCλ mutations (e.g., dominant gene mutations) can be studied usingtransgenic mice carrying mutated PKCλ transgenes or by introducing suchmutations into the endogenous PKCλ gene, using standard homologousrecombination techniques.

A replacement-type targeting vector, which can be used to create aknockout model, can be constructed using an isogenic genomic clone, forexample, from a mouse strain such as 129/Sv (Stratagene Inc., LaJolla,Calif.). The targeting vector can be introduced into a suitably derivedline of embryonic stem (ES) cells by electroporation to generate ES celllines that carry a profoundly truncated form of a PKCλ gene. To generatechimeric founder mice, the targeted cell lines are injected into a mouseblastula-stage embryo. Heterozygous offspring can be interbred tohomozygosity. PKCλ knockout mice provide a tool for studying the role ofPKCλ in embryonic development and in disease. Moreover, such miceprovide the means, in vivo, for testing therapeutic compounds foramelioration of diseases or conditions involving PKCλ-dependent or aPKCλ-effected pathway.

Use of PKCλ as a Marker for Stem Cells of the Heart

As PKCλ is expressed in cells that give rise to the heart during thecourse of development, it can be used as a marker for stem cells of theheart. For example, PKCλ can be used to identify, sort, or target suchstem cells. A pool of candidate cells, for example, can be analyzed forPKCλ expression, to facilitate the identification of heart stem cells,which, based on this identification can be separated from the pool. Theisolated stem cells can be used for many purposes that are known tothose of skill in this art. For example, the stem cells can be used inthe production of new organs, in organ culture, or to fortify damaged ortransplanted organs.

Experimental Results

Concentramide Specifically Modulates a Biological Pathway Involved inHeart Patterning

Zebrafish embryos have recently been shown to be amenable tohigh-throughput screening to identify small molecules that perturbdevelopmental processes (Peterson et al., Proc. Natl. Acad. Sci. U.S.A.97:12965-12969, 2000). In one such screen, we exposed developingzebrafish embryos to small molecules from a large, diverse chemicallibrary. Visual inspection of the transparent embryos was used toidentify small molecules that affect the global patterning of the heart.One of these small molecules is a biaryl compound containing anacrylamide moiety that we call concentramide (FIG. 1A), originallyidentified as library number 32P6 (Peterson et al., supra).

Normally, by 24 hours post-fertilization (hpf) the heart tube assemblesin the midline, with the atrium anterior to the ventricle and slightlydisplaced towards the left (FIG. 2A), and blood flow is driven fromatrial to ventricular end, first by persistalsis and then by sequentialchamber contractions. By 30 hpf, the chambers are clearly demarcated(FIG. 2B, using cardiac myosin light chain 2, cmlc2, to label bothchambers) and express different genes, as shown in FIG. 2C(ventricle-specific myosin heavy chain and atrial-specific antibodyS46).

Embryos exposed to concentramide develop compact hearts that do notsustain a circulation. It appears that both the atrium and ventricleform and beat in a coordinated manner in these fish, but that theventricle forms in the center of the atrium, as shown in FIGS. 2E and2F. The result is a heart in which the atrium and ventricle form twoconcentric rings, the inner ring composed of the ventricle and the outerring composed of the atrium. From the dorsal view, the heart looks likea bullseye (FIG. 2D), and from the lateral view, it looks like aninverted mushroom, in which the ventricle forms the stalk of themushroom and the atrium surrounds and covers the ventricle like amushroom cap (FIG. 1B).

Several observations suggest that concentramide is a highly specificmodulator of a particular molecular pathway critical to heartpatterning. Concentramide is very potent, with an ED₅₀ of about 2 nM.More importantly, higher doses of concentramide do not appear to causeadditional side effects. Concentramide causes virtually the samephenotype when used at a concentration of 6 μM as it does when used at aconcentration of 6 nM, suggesting that it modulates a specific moleculartarget at least 1,000 times more potently than it modulates otherproteins affecting visible developmental processes. The effect ofconcentramide on cardiovascular development does not appear to be aresult of general cytotoxicity. Development of concentramide-treatedembryos is not delayed relative to untreated siblings, and no increasein cell death is apparent. Concentramide also has no effect on the rateof proliferation of yeast or bromodeoxyuridine incorporation inmammalian cells. Given the potency of concentramide, its phenotypicreproducibility over a broad concentration range, and the rarity of thephenotype it produces (none of the >2000 other small molecules screenedgenerates a similar phenotype), we conclude that concentramide is aspecific modulator of a biological pathway responsible for heartpatterning.

A Time Window for Concentramide Effects

One advantage of small molecules over genetic mutations in studying adevelopmental process is that small molecules allow the process to bemodulated with much greater temporal control. Small molecules can beadded or washed away at any time during development, whereas geneticmutations are generally present throughout development. This temporalcontrol afforded by small molecules facilitates the identification ofcritical periods for developmental processes.

To identify the developmental stage at which concentramide disruptsheart patterning decisions, we added concentramide to the water ofdeveloping embryos at various times. As shown in FIG. 1C, embryostreated at any time prior to 14 hpf exhibit the concentric chambermorphology at 24 hpf, while embryos treated after 17 hpf exhibitwild-type heart morphology at 24 hpf. Repeating the experiment with moreprecise staging revealed that concentramide must be present before the14-somite stage (approximately 15 hpf) to induce the concentric chambermorphology. Therefore, a developmental event occurring at the 14-somitestage is critical for heart patterning and is disrupted by the smallmolecule concentramide.

The Hearts of Concentramide-treated Embryos Phenocopy Heart-and-soulMutants

Heart-and-soul (has) is a mutation isolated in our large-scale geneticscreen. The hearts of homozygous has mutant embryos are small. We findhere that, like those of concentramide-treated embryos, the hearts ofhas mutant embryos have ventricular tissue within the atrium (FIGS. 2Gand 2H). They manifest radial sequential contractions of the atrium,then the ventricle. The has mutant embryos, however, also manifestdefects in many tissues including the retina, kidney, gut, and brain.These defects are not present in concentramide-treated embryos. Thebrains of concentramide-treated embryos develop abnormally, but treatingembryos between 9 and 14 hpf eliminates this brain defect, whilepreserving the concentric heart chamber phenotype (FIG. 1C). Therefore,the heart phenotypes of concentramide-treated and has mutant embryos arevery similar, but concentramide-treated embryos appear to have fewerdevelopmental defects elsewhere, and the cardiac specificity of thephenotype can be increased further by controlling the timing ofconcentramide treatment.

Heart-and-soul Encodes an Atypical PKCλ

Given the phenotypic similarities between hearts from has andconcentramide-treated embryos, we reasoned that cloning the has genemight provide molecular insight about the process of heart patterning.Furthermore, cloning of has might allow us to determine whether has andconcentramide influence heart patterning through similar or distinctmechanisms. We mapped has by linkage analysis with zebrafish SSR markers(Michelmore et al., Proc. Natl. Acad. Sci. U.S.A. 88:9828-9832, 1991;Knapik et al., Nat. Genet. 18:338-343, 1998; Shimoda et al., Genomics58:219-232, 1999) and AFLP (Vos et al., Nucleic Acids Res. 23:4407-4414,1995) to an interval flanked by markers z8451 and z11023 ofapproximately 1.1 cM (FIG. 3A). These were used to initiate a walk usingYACs and BACs, which proceeded by end-cloning, refined mapping, andultimately sequencing. Genes identified as candidates for the mutationwere assayed by in situ analysis and for cDNA polymorphism by RT-PCR ofwild-type and mutant RNA pools. The genes contained within the BACs areshown in Table 1. The gene assignments are based on BLASTX alignments.

TABLE 1 Candidate genes identified within the heart-and-soul intervalBAC address identified genes (GenBank accession#) 109f10/122n17 KIAA0670protein/acinus (NP_055792) membane-type 1 metalloproteinase precursor(AAD13803) adaptin, gamma (NP_001119) KIAA1416 protein, novel HelicaseC-terminal domain and SNF2 N-termina domains containing protein, similarto KIAA0308 (CAB57836) ZPC domain containing protein 2 (AAD38907) zincfinger protein sal (AAB51127) cerebellin 1 precursor (NP_004343) RINGfinger protein (AAB05873) 152p21 unknown (NP_056541) 89i15precerebellin-like protein (AAF04305) 23c14 PKCλ transforming proteinsno-N - chicken (I51298) 53c17 no genes detected by BLASTX (mostlyrepetitive)

By sequencing PKCλ from wild type and mutant embryos, we confirmed thatboth has alleles harbor mutations in the PKCλ coding sequence. Themutation in the m567 allele causes a premature stop codon after aminoacid 518, and the mutation in the m129 allele causes a premature stopcodon after amino acid 514 (FIG. 3A). We determined the complete genomicstructure of the zebrafish PKCλ gene by shotgun sequencing of BAC 23c14.It is comprised of 18 exons spanning approximately 45 kb. We find PKCλmRNA to be expressed in a broad range of tissues.

The C-terminal truncation of PKCλ does not appear to destabilize theprotein, as truncated protein is detected by western blot analysis ofmutant embryos (FIG. 3B). However, truncation might be predicted toeliminate a domain essential for PKCλ function, given that C-terminaltruncation of PKCα or PKCβ renders these related kinases catalyticallyinactive (Riedel et al., J. Cell. Biochem. 52:320-329, 1993; Riedel etal., Mol. Cell. Biol. 13:4728-4735, 1993). In order to confirm the roleof PKCλ 20 mutation in the phenotype, we injected antisense morpholinooligomers complementary to the PKCλ translational start site. Theseinjections phenocopy the mutation entirely.

The injected embryos are indistinguishable at the gross morphologicallevel from the genetic mutants (FIG. 3C), supporting the idea that lossof the C-terminal 70 amino acids is sufficient to eliminate genefunction.

The Integrity of Epithelia is Affected by PKCλ Mutation, but not byTreatment with Concentramide

PKCλ belongs to the large PKC family of kinases and, with PKCζ, isclassified as an ‘atypical’ PKC (Mellor et al., Biochem. J. 332:281-292,1998). The presumptive ortholog of PKCλ in C. elegans, PKC-3,colocalizes with Par3 and Par6 at the anterior pole of the one-cellembryo (Tabuse et al., supra; Hung et al., Development 126:127-135,1999). PKC-3 is necessary for establishment of embryonic polarity, andinactivation of PKC-3 leads to mislocalization of the Par genes and asymmetrical first cell division. Drosophila possesses only one atypicalPKC (DaPKC), which also associates with a Par3-like protein (Bazooka)and is implicated in control of cell polarity (Wordarz et al., supra).DaPKC mutants exhibit disordered epithelial layering, irregular cellshapes, and loss of epithelial cell polarity, believed to be due todefects in cell adhesion. In vertebrate cells, PKCλ and PKCζ bothlocalize to epithelial tight junctions and associate with a Par3-likeprotein (ASIP) (Joberty et al., Nat. Cell Biol. 2:531-539, 2000; Suzukiet al., J. Cell Biol. 152:1183-1196, 2001; Lin et al., Nat. Cell Biol.2:540-547, 2000; Izumi et al., J. Cell Biol. 143:95-106, 1998). Wetherefore examined whether the has mutation and concentramide treatmentperturb epithelial patterning and tight junctions, focusing upon theretina and the kidney.

The neural retina arises from an epithelial sheet that is bordered bythe lens on the basal surface and by a second epithelial sheet (theretinal pigmented epithelium, RPE) on the apical surface (Schmitt etal., J. Comp. Neurol. 344:532-542, 1994). Prior to cell differentiation,the nuclei of the neuroepithelial cells migrate between the apical andbasal surfaces of the epithelium. During M-phase, cell nuclei localizeto the apical surface, adjacent to the neighboring RPE (Sauer, J. Comp.Neurol. 62:377-405, 1935). Beginning at about 30 hpf, theseneuroepithelial cells exit the cell cycle and differentiate into one ofseven distinct cell types (Altshuler et al., “Specification of Cell Typein the Vertebrate Retina,” In Development of the Visual System, Lam etal. (Eds.), The MIT Press, Cambridge, Mass. 37-58, 1991; Dowling, “TheRetina,” Belknap Press, Cambridge, Mass., 1987). Each cell type thenmigrates to a specific layer in the retina, resulting in a highlyorganized, laminar pattern (see FIG. 4A).

The has mutation causes disruption of the layering of the neural retinaand patchy loss of the RPE (FIG. 4E). These defects resemble those notedpreviously in zebrafish bearing the mutations oko meduzy (ome) andmosaic eyes (moe) (Jensen et al., Development 128:95-105, 2001; Malickiet al., Development 126:1235-1246, 1999). In has mutants, the severityof laminar disruption correlates with the position and degree of RPEdiscontinuity, suggesting that the RPE epithelial defect causes orexacerbates that of the neural retina. This would be concordant with theevidence that a normal RPE is critical to lamination (Raymond et al.,Curr. Biol. 5:1286-1295, 1995; Vollmer et al., Neurosci. Lett.48:191-196, 1984) and the fact that the retinal epithelium of hasmutants manifests at least one attribute of proper apical-basal polarityin that the majority of the mitotic nuclei localize correctly to theapical surface of the neuroepithelium (FIGS. 4B, 4D, and 4F; 89% ofM-phase nuclei from has embryos localize to the apical surface versus97% of nuclei from wild-type embryos). As a marker of tight junctions,we examined immunoreactive zonula occludens (ZO-1), an integral tightjunction protein, and find it to be mislocalized (FIGS. 4G and 4H).Therefore, loss of adhesion between RPE cells may be a cause of retinalmispatterning in has mutants. Notably, retinas fromconcentramide-treated embryos do not exhibit defects in cell polarity(FIG. 4D), RPE continuity, or lamination (FIG. 4C)

The developing kidney is another structure composed of highly polarizedepithelial cells. We examined the distribution of apical and basolateralproteins in the kidneys of wild-type, has, and concentramide-treatedembryos. As in the retina, cell polarity appeared to be largelyconserved in has kidneys (FIGS. 5A-5C). The has kidneys did, however,exhibit irregularities in the shapes of epithelial cells and occasionalgaps between cells, consistent with a defect in epithelial celladhesion. We did not observe these defects in embryos exposed toconcentramide.

Given the differences between has and concentramide-treated embryos withregard to epithelial sheet integrity in the retina and the kidney, it isunlikely that concentramide functions through the same mechanism as thehas mutation, namely the inactivation of PKCλ. To examine this further,we tested the effect of concentramide on early development of the C.elegans embryo. In C. elegans, inactivation of the PKCλ ortholog PKC-3via RNA interference (RNAi) results in the loss of polarizedlocalization of the Par proteins and loss of asymmetry during the firstcell division (Tabuse et al., supra). Embryos treated with highconcentrations of concentramide retain proper localization of Par2 tothe posterior pole and undergo a normally asymmetric first cell division(FIGS. 5D and 5E). Treated embryos exhibit cytolinetic defects and failto complete development, suggesting that the absence of an asymmetrydefect is not due to problems with compound penetration. Therefore,although concentramide treatment and PKCλ inactivation both result insimilar heart patterning phenotypes, concentramide does not appear toinactivate zebrafish PKCλ or its nematode ortholog.

The Molecular Target of Concentramide is Involved in AP Patterning

If the molecular target of concentramide does not affect the continuityof epithelial sheets as PKCλ does, by what sort of process might itinfluence heart patterning? Treatment with concentramide appears toaffect the relative positions of several anatomical structures along theanterior-posterior (AP) axis. For example, the distance betweenPax2.1-expressing cells in the eyes and at the midbrain/hindbrainboundary is reduced in concentramide-treated embryos (FIGS. 6A-6C).Perhaps more significantly, the cardiac myosin light chain 2(cmlc2)-expressing cells of the heart field are shifted rostrally inconcentramide-treated embryos at the 18-somite stage (FIG. 6D). Thedistance between the anterior edge of the cmlc2-expressing field and theanterior extreme of the embryo is about 40 percent greater in wild-typeembryos (3.1+/−0.2 arbitrary units, n=8) than in concentramide-treatedembryos (2.2+/−0.3 arbitrary units, n=12). The position of the heartfield in has mutants (3.1+/−0.3 arbitrary units, n=12) does not differsignificantly from the wild-type position. Therefore, the moleculartarget of concentramide appears to play a role in AP patterning.

PKCλ and the Target of Concentramide Both Influence the Fusion Order ofHeart Primordia

PKCλ and the molecular target of concentramide appear to act viadistinct cellular mechanisms, but modulation of either results in a verysimilar change in the patterning of the heart. To identify thecommonalties between the two mechanisms that allow such similarmispatterning of the heart, we took advantage of the temporal controlwith which small molecules can modulate biological processes. Asdescribed above, we determined that embryos must be treated withconcentramide at or prior to the 14-somite stage to cause formation ofthe ventricle within the atrium. From this observation, we conclude thata critical heart patterning process is initiated shortly after the14-somite stage, and perturbation of this process results in theconcentric chamber phenotype observed in both has andconcentramide-treated embryos. This allowed us to focus our search forcommonalties between has and concentramide-treated embryos to thiscritical time period.

The generation of the primitive heart tube is accomplished by midlinecoalescence of the bilateral cardiac primordial sheets. In thezebrafish, this coalescence first generates a single midline cone, withits base on the yolk (Fishman et al., supra; Yelon et al., Dev. Biol.214:23-37, 1999). Subsequently, the cone tilts to assume a midline A-Porientation with the pre-ventricular end posterior, later to swinganteriorly as yolk is resorbed.

We find that normally the generation of the midline cone does not occuruniformly around the cone's circumference, but rather progresses fromposterior to anterior, with posterior regions merging at the 16-somitestage and anterior at the 18-somite stage. This step is perturbed byboth concentramide and the has mutation. In both has mutant embryos andconcentramide-treated embryos, there is a failure to merge the posteriorends (FIGS. 7A-7C). Even by the 18-somite stage, when the anterior endsof the primordia begin to fuse normally, the posterior ends remainseparated in the has and concentramide-treated embryos (FIGS. 7D-7F).Eventually, the posterior ends do fuse in has and concentramide-treatedembryos, just before emergence of the concentric chambered heart. Thus,a critical patterning decision occurs at about the 16-somite stage thatregulates the fusion order of the anterior and posterior ends of theheart field. This process can be blocked either by inactivation of PKCλor by modulating the target of concentramide.

Thus, in summary, we have defined a key step in heart formation by itsperturbation with a small molecule and a mutation. This step involvesthe proper alignment of the two cardiac chambers, just as the primitiveheart tube assembles. Two perturbants—the small molecule concentramideand the has mutation—both elicit a previously undescribed chambermalalignment, in which the ventricle forms inside of the atrium. Thismeans that establishment of the cardiocyte cell fates is largelyaccomplished, but the higher order assembly of chamber structure isdisrupted.

Experimental Methods

Small Molecule Treatment

Zebrafish were maintained at 28.5° C. as described (Westerfield, “TheZebrafish Book, Guide for the Laboratory Use of Zebrafish (Daniorerio),” Univ. of Oregon Press, Eugene 1995). Unless specifiedotherwise, embryos were treated prior to gastrulation by addingconcentramide to the water at a final concentration of 34 nM from a 34μM stock solution in DMSO.

Whole-mount in Situ Hybridization and Immunohistochemistry

Digoxigenin-labeled antisense RNA probes were generated by ill vitrotranscription for cmlc2 (Yelon et al., supra), vmhc (Yelon et al.,supra), and pax2.1 (Krauss et al., Development 113:1193-1206, 1991). Insitu hybridization was carried out as described (Oxtoby et al., NucleicAcids Res. 21:1087-1095, 1993). For whole-mount immunohistochemistry,embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline(S46 and 3G8) or 80% methanol, 20% dimethyl sulfoxide (α-ZO-1),permeablized in acetone for 30 minutes at −20° C. (3G8), blocked with 5%fetal bovine serum, and incubated with the antibodies S46, 3G8 (Vize etal., Dev. Biol. 171:531-540, 1995), or α-ZO-1. An anti-mouse-horseradishperoxidase conjugate was used as secondary antibody for S46 and 3G8, andan Alexa 488-labeled anti-mouse secondary antibody was used for α-ZO-1staining.

Histology

Fixed embryos were dehydrated, embedded in plastic (JB4, Polysciences,Inc.), and sectioned at 2-7 μm. Retinal sections were stained withhematoxylin-eosin or dapi.

Cloning of Has

Embryos were separated into mutant and wild-type pools based onphenotypic analysis. Genomic DNA was isolated from individual embryos byincubation in DNA isolation buffer overnight at 50° C. (DNA isolationbuffer: 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 0.3% Tween-20; 0.3% NonidetP40; 0.5 mg/ml proteinase K). Proteinase K was inactivated prior to PCRsetup by heating samples to 98° C. for 10 minutes. PCR reactions wereperformed using diluted genomic DNA as described (Knapik et al.,Development 123:451-460, 1996).

RNA was isolated (RNeasy columns, Qiagen) from pools of wild-type andmutant embryos to generate cDNA for RT-PCR analysis (SMART RACE cDNAamplification kit, Clontech). Fragments were then subcloned intoPCRII-TOPO (Invitrogen). PCR primers were synthesized based on sequencefrom an EST for PKCλ (fc69h04, GenBank accession# AI883774) and genomicsequence (Genome Systems, BAC clone address 23c14), and used to sequencethe entire PKCλ coding region and 3′UTR.

Genomic clones were isolated by PCR analysis of DNA pools from BAC(Genome Systems) and YAC (Research Genetics) libraries using primer setsfor the linked markers z11023 and z8451. YAC end sequence was determinedas described (Zhong et al., Genomics 48:136-138, 1998). BAC ends weresequenced directly using SP6 and T7 primers, and BACs 53c17, 89i15, and152p21 were subcloned by shotgun cloning of partial AluI digestedfragments into pBluescript. For the complete sequencing of BACs, ahydroshear was used to produce fragments of 2-3 kb in length. Thesefragments were then blunt-end ligated into pGEM5 (Promega) and sequencedusing an ABI3700 to generate approximately five-fold coverage. Thesequence was assembled using the Phred/Phrap/Consed programs (Gordon etal., Genome Res. 8:195-202, 1998; Ewing et al., Genome Res. 8:186-194,1998; Ewing et al., Genome Res. 8:175-185, 1998).

Western Blotting

Groups of 25 embryos were lysed in 0.5% Triton X100 inphosphate-buffered saline. Lysates were clarified by centrifugation andseparted by 10% sodium dodecyl sulfate polyacrylamide gelelectrophoresis. Western blotting was performed using an α-PKCλ rabbitpolyclonal antibody (Santa Cruz Biotechnology, Inc.).

Morpholino Injection

An antisense morpholino oligonucleotide of sequence5′-CTGTCCCGCAGCGTGGGCATTATGG-3′ (SEQ ID NO:6) (GeneTools, LLC) wasdissolved at a concentration of 100 aM in 1× Danieau's buffer (5 mMHepes pH 7.6, 58 mM NaCl, 0.7 mM KCl, 0.6 mM Ca (NO₃)₂, 0.4 mM MgSO₄).One nL of this solution or 1× Danieau's buffer was injected into each1-4 cell embryo before allowing the embryos to develop at 28.50° C.

C. elegans Development

C. eleganis strain KK871 (par-2::GFP) was maintained at 25° C. For eachsample, 10-15 adult worms were soaked in 80 μL M9 medium containing 34μM concentramide, 0.25% dimethyl sulfoxide for 30-60 minutes. Worms werethen cut open with a scalpel, and embryos were mounted on 2% agarosepads with coverslips. Embryos were allowed to develop at 25° C. beforebeing photographed live.

Other Embodiments

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it is to be understood that it is capable offurther modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and can be applied to theessential features hereinbefore set forth, and follows in the scope ofthe appended claims.

1. A method of determining whether a human test subject has, or is atrisk of developing, a heart disease or condition related to ProteinKinase C λ, said method comprising analyzing a nucleic acid molecule ofa sample from the test subject to determine whether the test subject hasa mutation in a gene encoding human Protein Kinase C λ, wherein saidgene encoding said Protein Kinase C λ encodes the sequence of SEQ IDNO:5, and the presence of a mutation indicates that said test subjecthas, or is at risk of developing, a heart disease or condition relatedto Protein Kinase C λ.
 2. The method of claim 1, wherein said geneencoding human Protein Kinase C λ comprises the sequence of SEQ ID NO:4.3. The method of claim 1, wherein said heart disease or condition isassociated with epithelial-epithelial cell interactions or epithelialcell polarity.
 4. The method of claims 2, wherein said heart disease orcondition is associated with epithelial-epithelial cell interactions orepithelial cell polarity.
 5. The method of claim 1, wherein saidmutation results in a carboxyl terminal truncation of Protein Kinase Cλ.
 6. The method of claim 2, wherein said mutation results in a carboxylterminal truncation of Protein Kinase C λ.
 7. The method of claim 3,wherein said mutation results in a carboxyl terminal truncation ofProtein Kinase C λ.
 8. The method of claim 4, wherein said mutationresults in a carboxyl terminal truncation of Protein Kinase C λ.
 9. Themethod of claim 1, wherein said mutation is the heart and soul mutation.10. The method of claim 2, wherein said mutation is the heart and soulmutation.
 11. The method of claim 3, wherein said mutation is the heartand soul mutation.
 12. The method of claim 4, wherein said mutation isthe heart and soul mutation.
 13. The method of claim 5, wherein saidmutation is the heart and soul mutation.
 14. The method of claim 6,wherein said mutation is the heart and soul mutation.
 15. The method ofclaim 7, wherein said mutation is the heart and soul mutation.
 16. Themethod of claim 8, wherein said mutation is the heart and soul mutation.